Fabrication of nanoemulsion delivery system with high bioaccessibility of carotenoids from Lycium barbarum by spontaneous emulsification

Abstract The interest in incorporating carotenoids into foods and beverages is growing due to their potential health benefits. However, the poor water solubility and low bioavailability of carotenoids are still challenges in food application. This work aimed to study the influence of system composition and preparation conditions on the physical properties of carotenoids‐loaded nanoemulsions prepared by spontaneous emulsification. Furthermore, the bioaccessibility of carotenoids in the nanoemulsions was evaluated. The nanoemulsions with the smallest droplet size were produced when the ratio of Span 80:Tween 80 was 1.5:8.5. The droplet size increased slightly with the increase of organic phase content (24%–40%). The droplet size decreased gradually with the increase of stirring speed (200–1000 rpm (revolutions per minute)). The ratio of mixed surfactants and surfactant‐to‐oil ratio (SOR) had an appreciable impact on the droplet size. Carotenoids‐loaded nanoemulsions with small mean droplet size (d < 50 nm) could be prepared with the optimized conditions. The initial digestion rate decreased as the SOR increased. The bioaccessibility could reach up to about 80% at SOR=2–5 in vitro digestion. These results have important implications for the design of effective delivery systems to encapsulate carotenoids and other lipophilic bioactive components in food applications.

To overcome the limitations, a useful strategy is to encapsulate carotenoids into appropriate delivery vehicles. Various delivery systems have been developed to load lipophilic functional agents, including micelle, emulsions, colloids, biopolymer matrices, powders, and so on (McClements, 2013). Emulsion-based delivery systems are available to deliver bioactive lipids for the large interest in the development of nutraceutical or functional foods. Emulsions, nanoemulsions, and microemulsions are distinguished by their particle dimensions and thermodynamic stability (Mcclements, 2011).
Differences in particle dimensions lead to their different functional performances. The nanoemulsions with smaller droplets tend to have a higher stability to gravitational separation, flocculation, and coalescence than conventional emulsions. The droplets of nanoemulsions are so small that they only scatter light waves weakly. Therefore, they can be incorporated into optically transparent products without adversely affecting their clarity. Nanoemulsion-based delivery system can not only increase the solubility and stability, but also improve the bioavailability of lipophilic agents, which is considered an important target for nutrients delivery system (Huang et al., 2010).
The pH-stat method in vitro digestion has been widely used in food and pharmaceutical research to study the characterization of lipid digestion and bioaccessibility of active components . Previous studies have shown that the rate and extent of lipid digestion depended on numerous factors, including droplet size (Lee et al., 2011;Salvia-Trujillo et al., 2013;Troncoso et al., 2012;Yi et al., 2014), oil type (Lee et al., 2011;Salvia-Trujillo et al., 2013;Troncoso et al., 2012;Yi et al., 2014), lipid physical state (Bonnaire et al., 2008), and interfacial composition (Bonnaire et al., 2008). At the same time, the emulsions droplets could interact with various surface-active components, so the lipid digestion was also influenced by bile salts, pepsin, lipase, and minerals (Ca 2+ and Na + ). Previous results showed that emulsifier affected the absorption of pancreatic lipase onto the surface of emulsified fats (Mun et al., 2007). The effects of surfactants with low-molecular weight on the digestibility of lipids were further studied by Li. They found that the different types of surfactants have different inhibitions on lipid digestion (Yan et al., ). Low-molecular weight nonionic surfactants are the commonly used emulsifiers for the fabrication of nanoemulsions by spontaneous emulsification. However, the effects of low-molecular emulsifier content and mixing ratio on carotenoids-loaded emulsions droplet size, lipid digestion, and bioaccessibility have not been thoroughly studied.
In the paper, nanoemulsion systems were fabricated to encapsulate carotenoids from Lycium barbarum by spontaneous emulsification, aiming to improve the hydrophilicity and bioaccessibility of carotenoids. The influences of mixed surfactants ratio (Span 80:Tween 80), surfactants-to-oil ratio (SOR), organic phase content, and stirring speed on the physical properties of carotenoids-loaded nanoemulsions were studied. Furthermore, the influence of SOR on the lipid digestion and bioaccessibility of carotenoids in the nanoemulsions was investigated using the pH-stat method. The current study would have important implications for the design of effective delivery systems to encapsulate carotenoids and other lipophilic bioactive components in food or pharmaceutical applications.

| Fabrication of carotenoids emulsions
Emulsions were prepared by spontaneous emulsification using the method described previously with some modifications (Saberi et al., 2013). First, the surfactants (Tween 80 and Span 80) were mixed with MCT containing 0.2 wt.% carotenoids. The mixtures were taken as organic phase. Then, the organic phase was added dropwise into aqueous phase (distilled water containing 0.02 wt.% sodium azide) under magnetic stirring for 15 min at room temperature. In the preparation, the factors of Span 80-to-Tween 80 mass ratio, organic phase content, stirring speed, and surfactants-to-oil mass ratio were investigated.

| Droplet size and ζ-potential measurements
The average droplet size of the emulsions was determined by a Nano-ZS (Malvern Instruments). First, emulsions were diluted 100 times using 10 mM phosphate buffer solutions at pH 7.0 (PBS). Zaverage diameters for droplet size, polydispersity index (PDI), and ζ-potential were measured. The particle sizes of the digesta were also calculated using the surface-weighted mean diameters (d 3,2 ) by Mastersizer 2000 for its big dimension.

| Transparency measurements
The transparency of emulsions was measured at 600 nm using a spectrophotometer (UV-1100, meipuda instrument, China). Prior to the measurements, double-distilled water was used as a reference.

| In vitro digestion
The in vitro digestion was carried out by simulated small intestinal conditions, as described previously (Yan et al., ). The pH-stat (907 Titrando, Metrohm USA Inc.) was used to monitor and control the pH (pH 7.0) of the digestion process. Thirty milliliters of emulsions containing 0.5% MCT was placed into a bottle and then incubated in a water bath (37°C) for 10 min. Four milliliters of bile extract solution, containing 187.5 mg of bile extract (pH 7.0, PBS), was added into the 30 ml emulsions with stirring. Then, 1.0 ml of CaCl 2 solution and 1.0 ml of NaCl were added into the bottle with final concentrations of 10 and 150 mM, respectively. The pH value of the system was adjusted to 7.0 with NaOH solution. After 1.5 ml of freshly prepared pancreatin suspension (40 mg/ml, pH 7.0, PBS) was added, the automatic titration experiment was started immediately. Digestion experiments were performed for 120 min at 37°C. The amount of free fatty acids (FFAs) released during the digestion process was calculated from the following equation.
where C NaOH is the molarity of the NaOH solution used to titrate the sample (mol/L), V NaOH is the volume of NaOH solution required to neutralize the FFAs produced at digestion, and M oil is the molecular weight of the oil (g/mol). The molecular weight of the MCT was taken to be 500 g/mol, and m oil is the total mass of oil initially present in the incubation cell (g).

| Bioaccessibility determination
The bioaccessibility of carotenoids-loaded nanoemulsions was determined by the in vitro digestion process using a method described previously (Qian et al., 2012). An aliquot of the raw digesta was centrifuged at 16,000 rpm for 30 min at 4°C. The middle transparent layer was taken to be the ''micelle fraction'' in which the carotenoids were solubilized. Two milliliters of the micelle fraction was mixed with equivalent mixed solvent (n-hexane:ethanol = 1:1), vortexed, and centrifuged at 4000 rpm for 10 min at 25°C. The top layer with the dissolved carotenoids was collected, while the bottom layer was mixed with 2 ml of mixed solvent. The same procedure as before was followed until the bottom layer became colorless. The top nhexane layer was added to the previous one and the absorbance was measured at 450 nm using a spectrophotometer (UV-1100, meipuda instrument, China). Pure n-hexane was used as a reference. Total carotenoids content was calculated according to McBeth's formula as (Lin et al., 2011;Yuan et al., 2008).
where, absorbance (A) was determined by a spectrophotometer at 450 nm, V is the total volume of the solution (mL) and E 1cm 1% is 1% of the average extinction coefficient value of carotenoids in nhexane = 2500, and m is the weight of the sample (g). The carotenoids concentration of digesta was determined by the same method. The bioaccessibility was calculated using the following equation: where, C micelle and C digesta are the concentrations of carotenoids in the micelle fraction and in the overall sample (raw digesta) after the pHstat experiment, respectively.

| Statistical analysis
All measurements were performed in triplicate and were presented as means and standard deviations.

| Influence of the mixed surfactants ratio on the formation of nanoemulsions
It is widely known that hydrophilic-lipophilic balance ( type. An optimum surfactant's molecular geometry will promote the FFA released( % ) = 100 × C NaOH × V NaOH × M oil ∕ 2 × m oil .
Bioaccessibility( % ) = 100 × C micelle ∕C digesta F I G U R E 1 Effect of Span 80-to-Tween 80 ratio on (a) Z-average, polydispersity index (PDI) and (b) transparency of nanoemulsions. Nanoemulsions were prepared using 5 wt% oil, surfactants-tooil ratio (SOR) = 2, and stirring speed at 1000 rpm (revolutions per minute) spontaneous formation of small droplets at the oil-water boundary using low-energy methods (Guttoff et al., 2015). PDI values varied from 0.17 to 0.28 at all the ratios, which indicated the ratio of mixed surfactants had a slight impact on PDI. Figure 1b displays that the transparency of samples was higher at smaller particle size, and vice versa. The small droplet size compared with the wavelength of light means they tend to be transparent. Indeed, the transparency of nanoemulsions formed using these methods changed with the increasing droplet size, which was similar to the results reported in other studies (Mayer et al., 2013;Saberi et al., 2013).

| Influence of organic phase content and stirring speed on the formation of nanoemulsions
The impact of organic phase content on the droplet size of nanoemulsions was investigated ( Figure 2). The droplet sizes of nanoemulsions slightly increased from 23 to 38 nm, with the organic phase content increasing (Figure 2a). Because when SOR was kept constant at 3, the water content decreased with the increase of organic phase content in the system and led to the system viscosity increase.
The high viscosity does not favor the magnetic stirring and hinders the diffusion of organic phase to water phase during emulsification. Though the increase of droplet size was slight when the organic phase content was between 24% and 40%, the PDI obviously

| Influence of SOR on the formation of nanoemulsions
The influence of SOR on the formation of carotenoids nanoemulsions was investigated in this series of experiments. In these experiments, the total amount of MCT in the systems was kept constant (5 wt%), while the amount of surfactant was varied (5%-25%) with the remainder being buffer solution (90%-70%). The effects of SOR on the emulsions were investigated and the results are displayed in Figure 3. Increasing the SOR from 1 to 3 resulted in a significant decrease in the droplet size. A smaller droplet size means greater surface area, which requires more emulsifiers to F I G U R E 2 Effect of organic phase content (a, b) and stirring speed (c, d) on Z-average, polydispersity index (PDI) and transparency of nanoemulsions. Nanoemulsions were prepared using (a, b) Span 80-to-Tween 80 ratio 1.5:8.5, surfactants-to-oil ratio (SOR)=3, and stirring speed at 1000 rpm (revolutions per speed); (c, d) 5 wt% oil, Span 80-to-Tween 80 ratio is 1.5:8.5, and SOR = 2 cover. Furthermore, the increased adsorption of surfactant molecules to the oil-water interface leads to a decrease in the interfacial tension, which facilitates the formation of smaller droplets (Lamaallam, 2005). Other researchers have reported similar results that higher surfactant concentrations have produced smaller droplet sizes (Komaiko & Mcclements, 2014). The smallest droplets (about 20 nm) were formed at the SOR of 3, and the system was very transparent (Figure 3b). As SOR was higher than 3, the droplet size had a slight increase in trends, but the PDI increased dramatically from 0.17 to 0.59 (Figure 3a). It has been postulated that the droplet size increases above a certain surfactant level due to the formation of a highly viscous liquid crystalline phase, which makes spontaneous breakup of the oil-water interface more difficult (Mayer et al., 2013;Wang et al., 2009).

| Size and surface charge of emulsions after digestion
The with the values ranging from about −10 to −20 mV. When lipid droplets were exposed to simulated intestinal conditions that contained bile salts, which are anionic surface-active substances, the bile salts may displace the nonionic surfactants molecules from their surfaces and generate a negative charge Qian et al., 2012). In addition, the hydrolysis of neutral triglycerides by pancreatic lipase at the surface will lead to the release of anionic FFAs. Any of these molecules that remained at the droplet surfaces will also produce a negative charge. Different SORs do not cause much difference in the electrical characteristics of the particles in the digesta. It means that some or all the surfactants on the droplet surface have been replaced by bile salts or other molecules.

| Influence of SOR on lipids digestibility
The influence of SOR on the rate and extent of triglyceride digestion in the nanoemulsions using the pH-stat method was investigated ( Figure 5). The initial rate of lipid digestion decreased as the SOR increased from 1 to 5. At low SOR (≤3), the rate of lipid digestion was showed a much slower digestion rate. The results suggested that the lipid digestion was an interfacial phenomenon (Mcclements & Yan, 2010). The bulk lipids had a smaller surface area, which delayed the interaction with lipase.

| Effect of SOR on the bioaccessibility of carotenoids
The nanoemulsion systems showed a greater bioaccessibility than the bulk lipid sample ( Figure 6). The bioaccessibility of carotenoids dispersed in bulk lipid was only 2.4 ± 0.3%. Comparatively, the emulsions system showed a bioaccessibility of about 80% at SOR = 2-5. This phenomenon was related to the micellization process during digestion. When the bile salts were adsorbed onto the droplet surfaces and displaced any existing emulsifier molecules, the micelles were formed gradually with the carotenoids and products of digestion e.g. FFAs and MAGs. The bioaccessibility values at SOR = 1 were significantly different from those of other emulsions, which may be due to the relatively big droplet size (140 nm).
Generally smaller droplet size, i.e. higher surface area, was available for pancreatic lipases to attach more easily, resulting in an increased transfer of nutraceuticals to micelles (Yi et al., 2014).
Previous study also showed that the initial droplet size will affect the transfer efficiency of carotene (Pan et al., 2012). The results suggested that SOR will affect both droplet size and micelle formation. When SOR increased from 2 to 5, the bioaccessibility of carotenoids was similar owing to their small droplet size (20-60 nm).
However, some emulsifiers may be able to participate in the formation of micelles. Therefore, the actual metabolic pathway that carotenoids in nanoemulsions undergo in vivo digestion should be investigated for reasonable design of the nanoemulsion-based delivery system. Ideally, people would like to be able to form small droplets using the lowest amount of surfactant possible for economic, taste, and safety reasons. The result showed that nanoemulsions with small F I G U R E 3 Effect of surfactants-to-oil ratio (SOR) on (a) Z-average, polydispersity index (PDI) and (b) transparency of nanoemulsions. Nanoemulsions were prepared using 5 wt% oil, Span 80-to-Tween 80 ratio is 1.5:8.5, and stirring speed at 1000 rpm (revolutions per minute)

| CON CLUS ION
F I G U R E 4 Mean particle size (a) and ζ-potential (b) of nanoemulsions fabricated with different surfactant-to-oil ratios (SORs) before and after in vitro digestion F I G U R E 5 Influence of surfactantto-oil ratio (SOR) on lipids digestion and scheme of the emulsion droplets' interface droplet size and high bioaccessibility of carotenoids could be formed at SOR ≥2 (surfactant concentration ≥10%). The value is much higher than that of fabricated nanoemulsions using high-energy methods.
Self-emulsifying methods do not need expensive equipment and much energy, and only use simple stirring. Moreover, it is easy to fabricate fine nanoemulsions. The nanoemulsions can be diluted a lot of times and used in actual application to make it suitable for human consumption. Furthermore, in vivo studies would be useful to better understand the various physicochemical mechanisms that determine triglyceride digestion and the release of bioactive compounds from nanoemulsions system.

ACK N OWLED G M ENTS
This work was financially supported by the National Natural Science Dietology of Huazhong Agricultural University for offering many conveniences.

CO N FLI C T S O F I NTE R E S T
The authors declare that they do not have any conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.